Development of a Taqman Real-Time PCR Assay for Detection of Bordetella bronchiseptica
Development of a Taqman Real-Time PCR Assay for Detection of Bordetella bronchiseptica
Tomas Jinnerot1*, Karin Malm1, Erik Eriksson1 and Jonas Johansson Wensman2
E-mail | [email protected]
ABSTRACT
Infection with Bordetella bronchiseptica is the most common bacterial cause of kennel cough or canine infectious respiratory disease (CIRD) in dogs. Other disease agents include various respiratory viruses and co-infection can exacerbate the clinical signs. CIRD is highly contagious and can spread rapidly, especially among dogs in close quarters. Fast and accurate diagnostics enables the selection of proper management. Our objective was to develop a sensitive and specific real-time PCR for detection of B. bronchiseptica in samples from dogs with respiratory signs. A genome comparison program was used to select a suitable target DNA sequence. A PCR targeting the bfrZ gene was developed which showed no cross-reactivity in silico or when tested with a panel of bacterial isolates. The limit of detection was determined to 4x103 bacteria per mL of nasal swab sample and less than 10 copies of target DNA per PCR reaction. Out of 23 isolates of B. bronchiseptica tested, one isolate from a hedgehog was not amplified. Sequencing of the 16S rDNA showed that the isolate was similar to strains of both B. bronchiseptica and B. parapertussis but was considered to be of minor importance for the diagnostics of dogs. In a panel of culture-negative nasopharyngeal swabs from dogs with (n=57) or without (n=17) clinical signs of CIRD, three resulted PCR-positive for B. bronchiseptica. Altogether, the novel assay is highly specific and sensitive for detection of B. bronchiseptica in clinical samples.
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